1. Field of the Invention
The present invention relates to methods for studying tissue samples from a subject to predict the course of a tissue's tumor progression. In particular, the method determines the frequency of cells in a tissue which are undergoing abnormal cell division. More specifically, the invention relates to histochemical analysis of cell division in tissues.
2. Description of Related Art
The publications and other reference materials referred to herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference. For convenience, the reference materials are numerically referenced and grouped in the appended bibliography.
Cells often kill themselves in a process known as apoptosis or programmed cell death. Apoptosis is necessary for the body to properly function. Apoptosis is essential during embryonic development, differentiation, tissue remodeling, maturation of the immune system, tumor regression, and viral infection and is also involved in homeostasis. Faulty regulation of apoptosis may lie behind many diseases.
Both external stimuli and internal stimuli can induce apoptosis. For example, addition of glucocorticoid hormones to immature thymocytes, withdrawal of interleukin-2 from mature thymocytes, the removal of colony stimulating factors from haematopoietic precursors, and lack of adhesion in normal cells, may trigger apoptosis. Internal stimuli such as irreparable damage to genomic DNA or toxicity may also induce apoptosis. Cells undergoing apoptosis show a sequence of characteristic morphological changes including membrane budding, cytoplasmic shrinkage, and the condensation and margination of chuomatin. Frequently, apoptosis is associated with the activation of an endonuclease(s) that degrades the genomic DNA into large or oligonucleosomal fragments to yield a characteristic DNA ladder upon gel electrophoresis. The dying cells are broken into membrane-bound apoptotic bodies, which are phagocytosed by neighboring cells, avoiding a possible inflammatory response due to leakage of cell contents [reviewed in 1-6].
Apoptosis is often called programmed cell death because several gene products are involved in the regulation and execution of this process. An understanding of the set of genes involved in apoptosis comes from the studies on the nematode Caenorhabditis elegans [5]. These genes may fall into two groups: those that regulate the cell death program, and those that are involved in the execution phase of apoptosis. The nematode gene, ced-9, encodes a protein homologous to the mammalian Bcl-2 family of cell death regulators [7, 8].
The Bcl-2 family of apoptosis regulatory proteins consists of pro- and anti-apoptotic genes. In addition, the tumor suppressor genes p.sup.53 and Rb which are involved in cell cycle regulation are also involved in regulating apoptosis. Loss of their functions may confer resistance in tumor cells to apoptosis-inducing agents such as chemicals and ionizing radiations. Therefore, tumor cells are capable of surviving current therapeutic modalities, although the exact mechanism of tumor cell resistance to apoptosis-inducers is not well understood at present [9]. For prognosis, it is critically important to detect the earliest signs of tumor cells which have escaped anti-tumor therapy and are likely to progress toward neoplasia. Early detection methods are therefore needed.
The genes involved in the execution phase of apoptosis include the nematode gene, nuc-1, that encodes endonuclease required for DNA fragmentation, and ced-3, that encodes a cysteine protease with homology to interleukin-1.beta. converting enzyme (ICE)[10]. Recent studies have identified a protease, yama/CPP32.beta., as a mammalian homolog of CED-3 [11]. Other ICE-like family of enzymes include Nedd-2/Ich-1, Tx/Ich-2, and Mch-2 [reviewed in 6, 12].
Recent studies have shown that the morphological events characteristic of apoptosis can be induced in cytoplasts in the absence of a nucleus [13, 14]. Similarly, the cell nucleus was not required for cell-mediated granzyme- or fas-induced cell death pathways in target cells [15]. These and other studies have lead to the hypothesis that the gene products required for apoptosis are constitutively expressed in the cytoplasm [13, 16].
Although resistance of tumor cells to apoptosis inducers (e.g. chemotherapeutic agents and radiation) has been widely observed, the morphological changes of cells that escape anti-tumor therapy induced apoptosis and progress towards malignancy are unknown. There is a need to morphologically characterize the earliest stages of cells which escape induced apoptosis in order to predict the progression of the tumor cells in the patient toward malignancy or neoplasia.